Project Summary/Abstract: Mono-methylation of histone 3 lysine 4 (H3K4me1) is a chromatin mark closely associated with transcriptional enhancers and other intergenic regulatory elements; however, the functional significance of this histone modification has yet to be demonstrated. Our lab has previously identified Trr and MLL3/4 as the major H3K4- monomethylases in Drosophila and mammals, respectively. Recent genome-wide association studies identified the trr human orthologues, MLL3 and MLL4, as genes frequently mutated in a wide variety of human cancers, along with other COMPASS subunits. Previous work has established that Trr protein is necessary for regulating enhancer function; however, the role of Trr-dependent H3K4me1 at enhancers has not been tested directly. To address this, we have taken advantage of an embryonic recessive lethal Trr-NULL allele, trr[1], to test the necessity of Trr-dependent methylase activity. We have found the trr[1] lethality can be rescued by expressing a Trr transgene whose SET domain contains either a catalytic-inactive (C2398A) or a catalytic- hyperactive (Y2383F) point mutation. As expected, immunoblots confirm substantial reductions of H3K4me1 in the catalytic-dead mutant, while the catalytic-hyperactive mutation shows significant increases in H3K4me2/3. Our ChIP-seq studies verify these changes are occurring specifically at enhancer elements. Aside from these molecular phenotypes, the two mutant fly lines show no obvious developmental, reproductive, or behavioral differences in comparison with a control trr-WT rescue line. These observations raise three important questions: 1) what is the function of Trr-dependent H3K4-methylation in Drosophila development, 2) what are the non-enzymatic functions of Trr in regulating enhancer activity and transcriptional activation, and 3) how do mutations found in MLL3/4 disrupt enhancer function and lead to human cancer pathogenesis? By exploiting Drosophila melanogaster, in which MLL3/4 are represented by just one enzyme, trr, I will uncover fundamental details regarding how these proteins function to regulate enhancer activity, and how deleterious mutations to MLL3/4 result in cancer formation.